Vaccinia virus presents a model system in which to study virus multiplication, pathogenesis, and regulatory mechanisms. The usefulness of a genetic approach to study viral replication has been estalished by temperature-sensitive mutants of bacteriophage and more recently animal viruses. Because of the large size of the vaccinia genome (over 100 genes) a successful genetic analysis requires a large number of mutants in different genes as determined by complementation experiments and physiological characterization of their ts defects. In addition, the mutants must have low levels of reversion and leakiness. The primary aim of this research proposal is to isolate ts mutants of vaccinia virus and to determine their usefulness for genetic and biochemical studies according to the above criteria. After selection of an appropriate cell line and cloning of the wild-type at the nonpermissive temperature, virus will be mutagenized with 5-bromodeoxyuridine and nitrosoguanidine. Initially, plaques will be picked at random from monolayers infected with mutagenized virus at the permissive temperature and tested for the ability to produce plaques at the high and low temperatures. In later experiments, selection techniques will be employed to increase the utilization of rapid qualitative complementation techniques to expedite these experiments will be investigated. Finally, as time allows, the temperature-sensitive defects of the mutants will be investigated by examining the following aspects of viral replication at the nonpermissive temperature: viral undercoating, DNA replication, virus-induced polypeptide and antigen synthesis, induction of viral enzymes, inhibition of host macromolecular synthesis, and assembly of mature virions. In addition, the thermostability of mutant virions and virion-associated enzymes will be examined.